The present invention relates to a device for monitoring the activities of living cells in tissue culture using electronic means.
Some aspects of electrical monitoring of cells in culture have been disclosed by the present inventors in the scientific literature. Many of the capabilities and potential applications of this technique in medicine and biotechnology, have not been disclosed however. The inventors' earlier work which has ultimately led to the present invention is disclosed in Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 3761-3764, June 1984; IEEE TRANSACTIONS OF BIOMEDICAL ENGINEERING, Vol. BME-33 No. 2, February 1986; Physica D38 (1989) 128-133; and Journal of Immunological Methods, 127 (1990) pp. 71-77.
These articles do not disclose multiple electrode arrays that conform to more conventional tissue culture practice. Such electrode arrays would allow one to easily make measurements of several individual tissue cultures. Such multiple measurements would find use in many areas including in vitro toxicology, drug discovery, and drug efficacy measurements. These publications also do not disclose the use of a range of applied AC frequencies to obtain information regarding the spaces between the cell and substratum and the permeability of confluent cell layers.
Currently, electrical devices are known for measuring some activities of cells in culture. The best known of these measures resistance or porosity of cell sheets in culture. These measurements can only be applied to highly confluent and organized layers of epithelial and sometimes endothelial cells. Recently Molecular Devices Corporation (Palo Alto, Calif.) has introduced a cell microphysiometer that electrically follows the pH in the microenvironment about a few thousand cells. To be effective this device must be used in special medium that lacks buffers--a serious drawback for many applications.
Most animal cells in culture will only multiply when they are attached to a surface and are provided with the necessary nutrient medium and environment. Usually this surface or substratum is glass or specially treated polystyrene. Proteins from the medium will spontaneously absorb to these substrata and hence the surface encountered by the cells is always protein coated. The tissue culture is started or seeded when a cell suspension is added to a petri dish or other vessel, placed in a warm (37.degree. C.) environment and left undisturbed.
As suspended cells settle onto the protein-coated surface, they form attachments to the absorbed proteins over a period of about 30 minutes. Following this, a striking series of changes takes place in the cell's morphology. Attached to a substratum, the cells begin a process referred to as spreading as they flatten and extend themselves out on the surface. In time, they go from spheres of approximately 20 micrometer diameter to flattened forms with dimensions as large as 100 micrometers in the plane of the substratum and a thickness of only a few micrometers. This dramatic change in the cell's shape takes place over a period of a few hours dependent upon the type of cell and the protein that coats the substratum. Once anchored, cells display another remarkable behavior. By extending portions of the cell cytoplasm forward and away from the main cell body, making an attachment and then pulling the back of the cell up, the cell crawls about on the surface with a speed of a few micrometers per hour. Motility in tissue culture is found for almost all animal cells. These motions are generated in an outer band of cytoplasm and involve the cytoskeleton of the cell, in particular muscle-like strands of the protein actin referred to as microfilaments. This phenomenon which is observed in vitro is thought to be an expression of a basic cellular mechanism involved in processes including wound healing, maintenance of cellular organization in tissues, surveillance for invading foreign organisms, and development of the early embryo.
If conditions are suitable, the cell will copy its chromosomes in preparation for cell division. In mitosis, the highly spread cell retracts from the surface and again becomes spherical in morphology. When mitosis is complete, the cell splits into two spherical daughter cells. These new cells now initiate the spreading process and in a few minutes spread out again upon the substratum into a highly flattened morphology and the cycle begins anew. Tissue culture is an indispensable research tool. In addition, it has played an essential role in the development of modern biotechnology. Some of the powerful new applications are: pharmaceuticals, monoclonal antibodies, vaccines, genetic screening, skin grafts, gene therapy and in vitro toxicology.